The invention is directed to assays for determining the presence, activity, or both, of kinases and phosphatases. The preferred method is an assay for lipid kinases, phospholipid kinases, and phospholipid phosphatases.
The importance of phospholipids in general, and phosphoinositides in particular, in the regulation of cellular processes such as cell proliferation, apoptosis, and secretory functions has been recognized for a number of years. While the importance of these compounds is manifest, there is much that remains unknown about how these important cell-signaling compounds are regulated.
Phosphoinositides have the general formula shown in 1:
where each R is an unsubstituted or substituted alkyl, alkenyl, alkylcarbonyl, or alkenylcarbonyl group and R2, R3, R4, R5 and R6 are hydrogen atoms and their locations on the inositol moiety. These hydrogen atoms can be replaced by phosphate groups singly or in various combinations. The general structure 1 shall be referred to herein as a phosphoinositide, or simply a xe2x80x9cPI.xe2x80x9d Where relevant, the presence and position of phosphate groups on the inositol moiety will be designated by a number designation followed by the letter xe2x80x9cP.xe2x80x9d So, for example, a PI with a phosphate in the R3 position (i.e., phosphatidylinositol-3-phosphate) will be designated xe2x80x9cPI3P,xe2x80x9d phosphatidylinositol-4-phosphate will be designated xe2x80x9cPI4P,xe2x80x9d etc. For multiply phosphorylated PI""s, numbers separated by a comma will be used to designate the positions of the phosphate groups. For example, xe2x80x9cPI3,4P2xe2x80x9d designates a PI which is phosphorylated at the R3 and R4 positions of the inositol moiety as shown in 1. Phosphorylated PI""s in general are referred to as xe2x80x9cPIP""sxe2x80x9d
Because PI""s are thought to be central to signal transduction and membrane trafficking in all eukaryotes (Rao et al. (1998) Cell 94:829), an understanding of the enzymes that regulate PI""s, PIP""s, and their metabolites would be extremely helpful. The present invention, an assay to determine the presence and/or activity of lipid kinases, phospholipid kinases, and phosphatases can be used to elucidate further the biological role of PI""s.
Any number of assays for measuring enzyme activity are known in the prior art. In particular, Huang et al., U.S. Pat. No. 5,869,275, issued Feb. 9, 1999, describes an affinity ultrafiltration-based assay for measuring protein transferase activity. In this approach, labeled and unlabeled substrate having a binding site (the binding site either being created by action of the enzyme being assayed or which exists as an integral part of the substrate, such as an antigenic determinant) are incubated with the enzyme to form labeled product and unlabeled product. The reaction mixture is then contacted with a soluble macroligand capable of forming a specific complex with the product. Of critical importance is that the size of the macroligand-product complex must be significantly larger than the size of any contaminants or reactants found in the reaction mixture
The macroligand-product complex is then separated from reactants via ultrafiltration. The critical consideration here is that the nominal molecular weight limit of the ultrafiltration membrane must be larger than any potential contaminants in the reaction mixture, as well as larger than any unreacted, labeled substrate, yet smaller than the size of the macroligand-product complex. In this fashion, reactants and contaminants pass through the membrane, while the much larger macroligand-product complex is retained by the membrane. Examination of the ultrafiltration retentate for the presence of labeled product provides an indication of the extent of the reaction.
Mallia, U.S. Pat. No. 5,527,688, issued Jun. 19, 1996, describes assays for protein kinases in which a binding membrane is suspended within a reaction vessel, thereby dividing the vessel into two compartments. By adhering products to the suspended membrane, washing can be accomplished centrifugally by placing the wash solution in the upper chamber and centrifuging, thereby forcing the wash solution through the membrane.
The invention is an assay and a corresponding kit for determining the presence and activity of kinases and phosphatases, and, more specifically, lipid and phospholipid kinases and phosphatases. The preferred embodiment of the invention is an assay capable of measuring the presence and/or activity of any kinase or phosphatase which adds or removes a phosphate group from a lipid or phospholipid substrate.
In particular, a first embodiment of the invention is directed to a method for assaying the presence, the activity, or both the presence and the activity, of an enzyme falling within the enzyme classifications EC 2.7.1, EC 3.1.3, and EC 3.1.4. The method comprises first reacting the enzyme with a corresponding substrate for a time sufficient to yield phosphorylated product when assaying a kinase or a dephosphorylated product when assaying a phosphatase. The reaction, of course, is run under conditions which render the enzyme under investigation active, and thus the enzyme will catalyze either a phosphorylation (kinase) or a dephosphorylation (phosphatase) of the substrate.
The product formed by the enzymatic reaction is then contacted with a binding matrix. This results in product being bound or fixed to the matrix. With the product fixed on the matrix, the matrix can be mechanically separated from the reaction solution, thereby providing an easy means to separate the products of the enzymatic reaction from unreacted reactant, enzyme, and other non-product ingredients of the reaction solution.
The matrix is then analyzed for the presence of, the amount of, or both the presence and the amount of, the product fixed to the matrix. By determining the presence and/or amount of the product found on the matrix, the presence, the activity, or both the presence and activity of the enzyme that gave rise to the products can be determined.
A second embodiment of the invention is directed to a method as substantially described above, with the addition that the substrate includes a binding moiety. When the substrate is converted into product (by the action of the enzyme), the product also contains the binding moiety.
The product is then contacted with a binding matrix that specifically binds for the binding moiety. This results in product being specifically fixed to the matrix (via the interaction of the binding moiety and the binding matrix. The preferred binding moiety is biotin and the preferred binding matrix is avidin or streptavidin immobilized on an inert support. The matrix is then analyzed as in the first embodiment.
In the second embodiment of the invention, the approach used to modify the lipid or phospholipid enzyme substrate is to include a binding moiety. In this embodiment, the binding moiety (and the binding moiety alone) will bind specifically to a support designed for that purpose. In this fashion, products bearing the binding moiety can be separated easily from products which do not bear the binding moiety by simply passing the reaction mixture over the support. It is much preferred that the binding moiety be biotin. The support would then comprise any suitable substrate (beads, filter paper, etc.) having avidin or streptavidin immobilized thereto.
In a third embodiment of the invention, a support is not required. In this approach, the assay is conducted entirely in the liquid phase. Using a biotin binding moiety as an example, the avidin or streptavidin would be added to the reaction and the biotin-avidin complexes which form could be separated by any known means (electrophoresis, chromatography, centrifugation, etc.).
In short, the assay of this embodiment functions to determine the presence and activity of lipid and phospholipid kinases as follows: A lipid or phospholipid substrate for the enzyme to be assayed is first modified to include a binding moiety. As noted above, the binding moiety is preferably biotin, although an antibody or antigen can also be used as the binding moiety. Regardless of the choice of binding moiety, it is important that the binding moiety be attached to the substrate in such a fashion that its presence does not interfere with the enzyme""s ability to phosphorylate or dephosphorylate the substrate. In most instances, this requires that the binding moiety be added to the end of one of the fatty acyl moieties of the substrate because recognition is dictated largely by the nature of the headgroup.
To assay for lipid and/or phospholipid kinases, the modified substrate is exposed, in the presence of xcex3-32P ATP, to a solution thought to contain the kinase of interest and allowed to incubate for a sufficient amount of time and under appropriate conditions such that the kinase, if present and active, can phosphorylate the modified substrate with a 32P-labeled phosphate group. The reaction mixture is then contacted with a capture membrane or matrix, that is, a support bearing a moiety which will capture specifically the modified substrates; in the case of biotin, this would be avidin or streptavidin linked to a support. If binding moiety is an antigen, the capture membrane would include immobilized antibodies specific for the antigen, etc.
When the modified substrates are captured to a solid support, free 32P ATP, reactants, contaminants, etc., are gently washed from the support and the bound radiolabeled material is measured for radioactivity using a scintillation counter, a xe2x80x9cPhosphoImagerxe2x80x9d device, or by autoradiography.
Where phosphatases are to be assayed, the assay protocol is the same as noted above, with the exception that the substrate is modified to include xcex3-32P phosphate groups. In addition, an unlabeled substrate can be used and the released phosphate determined with a colorimetric method or a fluorescent method. (For example, Molecular Probes, Eugene, Oreg., sells a method for fluorescent detection of a released phosphate.) The action of the phosphatase enzyme under analysis will then remove a portion of those groups. In this approach, the reduced activity found on the matrix as compared to the starting labeled substrates is a direct measure of the activity of the phosphatase, or, in the case of non-radiolabeled phosphate, the amount of released phosphate is used as a measure of phosphatase activity.
The invention also comprises a corresponding kit that contains all of the necessary reagents to carry out the assay one or more times. The kit generally comprises: an amount of reaction buffer disposed in a first container; an amount of substrate for an enzyme classified within an enzyme classification selected from the group consisting of EC 2.7.1, EC 3.1.3, and EC 3.1.4, the substrate disposed in a second container; an amount of purified enzyme classified within an enzyme classification selected from the group consisting of EC 2.7.1, EC 3.1.3, and EC 3.1.4, the enzyme disposed in a third container (or the enzyme can be supplied by the user); an amount of binding matrix; and instructions for use of the kit.
A distinct and primary advantage of the assay is that is allows investigators to assay the activity of any number or type of lipid or phospholipid kinases or phosphatases quickly and conveniently.
Another primary advantage of the assay is that it is sufficiently sensitive to assay lipid and phospholipid kinases and phosphatases directly from tissue and cell extracts. Due to the high affinity of the lipid and phospholipid products to the matrix, extraneous free xcex332P-ATP can be removed by washing and the amount of product formed can be determined without need for lipid extraction. This is a distinct improvement over conventional HPLC and TLC assays, which require lipid extraction. Eliminating lipid extraction, which is costly and time-consuming, makes the subject assay very attractive to investigators in this field.
Another advantage is that the assay is scalable to accommodate high throughput formats. The assay is highly amenable to automation.